Investigating the dynamics of interchromosomal interactions and CTCF site methylation at the IGF2 locus in mammalian evolution and human disease

Abstract

Long-range physical interactions between distant sections of DNA have been shown to form complex networks of loops controlling gene regulation and other nuclear functions, which are essential throughout development and disease. These chromatin interactions are remarkably frequent, with interaction patterns varying between cell types, developmental stage and in disease. The chromatin insulator CTCF mediates many of these interactions, and is also thought play a role in the definition of topological domains and preventing the spread of heterochromatin. Binding of the CTCF protein can be methylation sensitive, and few studies have investigated the impact of specific methylation changes at CTCF binding sites on long-range interactions at a particular locus. This form of regulation is particularly important to many imprinted genes, which are important for foetal growth and development, such as the growth factor IGF2. Altering the regulation at this locus can affect foetal development and has also been shown to be linked to poor prognosis in several cancers. The aim of this project was to investigate the important IGF2/H19 locus in relation to long-range interaction and CTCF binding site methylation, in both developmental and disease contexts. We investigated expression of IGF2 and H19 as well as the frequency of long range chromatin interactions at the locus in cattle embryos, comparing purebred and hybrid crosses with known differences in birthweight. This work identified different levels of H19 expression between the different crosses, although no significant difference was observed in the frequency of the IGF2/H19-WSB1 long-range chromatin interaction. We have suggested that a different mechanism of regulation at the IGF2/H19 locus may occurring at this early developmental stage. We also investigated the methylation status of seven CTCF binding sites in the Igf2/H19 imprinting control region in several ovarian cancer tumours and cell lines, as well as looking at expression of key genes and interaction frequency using DNA Fluorescence in situ hybridisation. We identified highly variable DNA methylation patterns at CTCF binding sites in serous ovarian cancer tumours at different disease stages and noted that methylation at each site responded with variable sensitivity to treatment with a common demethylating drug in ovarian cancer cell lines.